Synthesis of 4′-Substituted Carbocyclic Uracil Derivatives and Their Monophosphate Prodrugs as Potential Antiviral Agents

Over the past decades, both 4′-modified nucleoside and carbocyclic nucleoside analogs have been under the spotlight as several compounds from either family showed anti-HIV, HCV, RSV or SARS-CoV-2 activity. Herein, we designed compounds combining these two features and report the synthesis of a series of novel 4′-substituted carbocyclic uracil derivatives along with their corresponding monophosphate prodrugs. These compounds were successfully prepared in 19 to 22 steps from the commercially available (-)-Vince lactam and were evaluated against a panel of RNA viruses including SARS-CoV-2, influenza A/B viruses and norovirus.


SARS-CoV-2 Antiviral Assays
The anti-SARS-CoV-2 activity of compounds herein prepared was evaluated at 10 μM following previously reported methods [20]. Briefly, a monolayer of Vero cells in a 96-well cell culture microplate was treated with 10 μM of each compound for 1 h followed by infection with SARS-CoV-2 (Washington Strain) at 0.1 MOI. After 1 h adsorption at 37 °C, the virus inoculum was removed, and the compound or vehicle-containing medium was added to the respected wells. Resultant virus progeny yield was measured 2 days (7) [18] were shown to display weak activities against a panel of RNA viruses including yellow fever (YF), dengue virus (DENV), venezuelan equine encephalis virus (VEE) and west nile virus (WNV), while 4′-ethynyl and 4′-cyano carbocyclic-2′-deoxyribonucleoside analogs (8a) and (8b) (Figure 2) displayed anti-HIV-1 activity [19] (Figure 2).  Based on these precedents, we wish to report herein the synthesis of novel 4′-substi tuted carbocyclic uridine analogs (9)(10)(11) and their corresponding monophosphate pro drugs (12)(13)(14) and report their evaluation against a small panel of RNA viruses including Norovirus, Influenza A, Influenza B viruses and SARS-CoV-2 ( Figure 3).

SARS-CoV-2 Antiviral Assays
The anti-SARS-CoV-2 activity of compounds herein prepared was evaluated at 10 μM following previously reported methods [20]. Briefly, a monolayer of Vero cells in a 96-well cell culture microplate was treated with 10 μM of each compound for 1 h followed by infection with SARS-CoV-2 (Washington Strain) at 0.1 MOI. After 1 h adsorption at 37 °C, the virus inoculum was removed, and the compound or vehicle-containing medium was added to the respected wells. Resultant virus progeny yield was measured 2 day Based on these precedents, we wish to report herein the synthesis of novel 4 -substituted carbocyclic uridine analogs (9)(10)(11) and their corresponding monophosphate prodrugs (12)(13)(14) and report their evaluation against a small panel of RNA viruses including Norovirus, Influenza A, Influenza B viruses and SARS-CoV-2 ( Figure 3).

SARS-CoV-2 Antiviral Assays
The anti-SARS-CoV-2 activity of compounds herein prepared was evaluated at 10 μM following previously reported methods [20]. Briefly, a monolayer of Vero cells in a 96-well cell culture microplate was treated with 10 μM of each compound for 1 h followed by infection with SARS-CoV-2 (Washington Strain) at 0.1 MOI. After 1 h adsorption at 37 °C, the virus inoculum was removed, and the compound or vehicle-containing medium was added to the respected wells. Resultant virus progeny yield was measured 2 days

SARS-CoV-2 Antiviral Assays
The anti-SARS-CoV-2 activity of compounds herein prepared was evaluated at 10 µM following previously reported methods [20]. Briefly, a monolayer of Vero cells in a 96-well cell culture microplate was treated with 10 µM of each compound for 1 h followed by infection with SARS-CoV-2 (Washington Strain) at 0.1 MOI. After 1 h adsorption at 37 • C, the virus inoculum was removed, and the compound or vehicle-containing medium was added to the respected wells. Resultant virus progeny yield was measured 2 days posttreatment from the supernatant of treated infected cells by specific quantitative RT-PCR.

Norovirus Antiviral Assays
The anti-NoV activity of compounds herein prepared was evaluated at 10 µM following previously reported methods [21,22]. Briefly, HG23 replicon cells, kindly provided by Kyeong-Ok Chang, Kansas State University (Manhattan, KS, USA), were seeded at a density of 1.6 × 10 4 cells/well in 96-well plates and incubated at 37 • C and 5% CO 2 overnight. Compounds were tested at 10 µM. Compounds were added in triplicate to 80-90% confluent monolayers and incubated at 37 • C and 5% CO 2 . Untreated cells were incubated in each plate. At 24, 48, 72 and 96 h post-treatment, total RNA was extracted using the Mag-Max Total RNA Isolation kit (Ambion, Austin, TX, USA) and NV replicon RNA was quantified by GI NoV Taqman real-time RT-PCR (NoV RT-qPCR). Protein expression levels were monitored by western blot analysis.

Influenza A/B Antiviral Assays
The anti-Influenza A/B activity of compounds herein prepared was evaluated at 40 µM following previously reported methods [23]. Briefly, A549 cells were seeded at a density of 35,000 cells/well and incubated at 37 • C and 5% CO 2 overnight. Cells were treated with test compound at 40 µM, then incubated at 37 • C and 5% CO 2 for 1 h before being inoculated with 0.1 MOI (PR8-PB2-Gluc) or 1.0 MOI (Ya88-PB1-NanoLuc).

Cytotoxicity Assays
The cytotoxicity of the compounds was evaluated using previously reported methods assays [24]. Briefly, in vitro cytotoxicity was determined using the CellTiter 96 nonradioactive cell proliferation colorimetric assay (MTT assay, Promega, Madison, WI, USA) in primary human peripheral blood mononuclear (PBM), human T lymphoblast (CEM), human hepatocellular carcinoma (Huh7) and kidney epithelial (Vero) cell lines. Toxicity levels were measured as the concentration of test compound that inhibited cell proliferation by 50% (CC 50 ).
In order to express their therapeutic effect, nucleoside analogs must be phosphorylated to their corresponding 5 -triphosphate forms by three different kinases. Interestingly, the first phosphorylation is often the limiting step in this process and several monophos-phate prodrugs that can be cleaved intracellularly to deliver the monophosphate form of a nucleoside have been developed [28]. Among them, phosphoramidate prodrugs (Protides) as seen in approved drugs such as sofosbuvir (HCV), tenofovir alafenamide (HIV), or remdesivir (SARS-CoV-2) have been well studied and their use has been validated clinically. Thus, the corresponding monophosphate prodrug of nucleosides (12)(13)(14) were prepared through the chemistry described in Scheme 3. Compound (9) was first protected as a 2 -,3 -acetonide intermediate (25) in 64% yield, before reacting it with isopropyl ((S)-(perfluorophenoxy)-(phenoxy)phosphoryl)-L-alaninate [29] (Reagent B) in presence of t-BuMgCl. Final deprotection under acidic conditions gave prodrug (12) in 34% yield over 2 steps. On the other hand, prodrugs (13) and (14) were prepared in 13% and 5% yields respectively, directly from the corresponding parent nucleosides (10) and (11) by reaction with Reagent B in presence of t-BuMgCl at 0 • C overnight. It is worth noting that, the protection of the 2 -3 -positions in compounds (10) and (11) (as described above for the synthesis of prodrug (12)) to form the corresponding prodrugs did not improve the overall yield as both 2 ,3 -isopropylidene monophosphate prodrugs appeared to be unstable under deprotection conditions.

Conclusions
A series of novel 4 -substituted carbocyclic uracil derivatives containing 4 -ethynyl, 4 -cyano and 4 -chloromethyl groups were synthetized in 19 to 22 steps from the (-)-Vince lactam and evaluated against a small panel of clinically relevant RNA viruses. Unfortunately, none of these compounds, nor their corresponding monophosphate prodrugs, displayed significant antiviral activity against SARS-CoV-2, IFV-A, IFV-B or norovirus. It is worth noting though that none of them showed toxicities up to 100 µM in a panel of cell lines including PBM, CEM, Vero and Huh7 cells.